1. Field of the Invention
The invention concerns a device for the application of post-column (derivatization) reactions (PCR) in liquid chromatography using a universally applicable device for the pulsation-free addition of reagents.
2. Description of the Related Art
Liquid chromatography (LC) is a highly sophisticated analytical separation technique. In general this term is understood as High-performance (High-pressure) liquid chromatography (HPLC). Using high pressure the solutes of a sample are separated on a column filled with special materials of small particle size (stationary phase) using a liquid solvent as mobile phase. The small particle size results in a high number of theoretical plates per unit of column length and provides excellent conditions for the separation of the solutes. The resulting high cross-sectional resistance, however, requires the use of powerful pumps to provide the force to drive the mobile phase through the column. These pumps are designed to cause as little pulsation as possible of the mobile phase. The detection of the solutes separated is performed using various procedures such as UN/VIS-, fluorescence-, conductivity- or electrochemical-detections as well as radioactivity monitoring systems after supplementation of the mobile phase with a scintillation fluid for the determination of solutes labeled with radioactive isotopes.
The application of this methodology is limited, however, by the general weakness of the detection systems. Although the diversity of these systems appears to be enormous, the detection of very low concentrations of analytes, which are often encountered in HPLC-analysis, is possible only in the case of specific chromophores and fluorophores. In particular, some of the very important groups of substances like sugars, amino acids and steroids etc. escape detection at low concentrations. Further, the lack of a generally applicable detection system, like the FID in Gas Chromatography, can be considered as a general disadvantage of Liquid Chromatography as an analytical tool.
One of the answers to these problems is the application of chemical derivatization methods. These methods are based primarily on chemical reactions resulting in the conversion of the analytes into chromophores or fluorophores providing an amelioration of the sensitivity and, in addition, a high degree of specificity since only the compound of interest is derivatized. Because of these advantages chemical derivatization has found broad acceptance as an analytical tool in other areas of chromatography.
In principle, two options exist for the application of chemical derivatization in HPLC-analysis, i.e. derivatization of the analyte before application of the sample material to the column (pre-column derivatization) or derivatization of the analyte after its elution from the column (post-column derivatization). In most cases, depending on the specific analytical problem, only one of these methods can be applied. The pre-column derivatization is well known and in general use. One of the advantages of this procedure is the fact that additional pumps are not required; further, it allows long reaction times and it can be used, if reagent and derivative have similar light absorption properties. The disadvantages resulting from pre-column chemical derivatization are changes of the separation characteristics of the analyte and the tendency of artifact development.
In many situations, however, post-column derivatization--from now on called "PCR" (post-column reaction)--has to be applied. This procedure is used preferentially in the "on-line" mode. The only prerequisite for the application of PCR is that the chemical derivatization reactions can be carried out in a reproducible manner; i.e.--although favorable--the reactions do not even have to go to completion prior to reaching the detector system nor do they have to be defined chemically. The advantages offered by the PCR procedure in selectivity of reaction and specificity in the subsequent detection are won at the expense of the sensitivity of detection; i.e. the theoretically possible detection limits of the available detection systems worsen under PCR-conditions by a more or less pronounced broadening of bands, which depends of the dimension and the quality of the PCR reactor used. The main cause of the loss of sensitivity is due, however, to a lack of stability of the background signal, which may cause a severe deterioration of the signal/noise ratio, the decisive criterion of the limits of analyte detection.
The various parameters, which are decisive determinants of the detection limit of a typical PCR analysis can summarized as follows:
1. The intensity of analytical signal PA1 2. The noise of the detector (high frequency noise) PA1 3. The stability of the background signal (low frequency noise) PA1 1. Syringes of any dimension can be used, provided they are resistant to the pressures encountered. A syringe of 50 ml capacity, for example, is sufficient to carry out analyses continuously over an 8 hour period assuming an infusion rate of 0.1 ml/min of the reagent and a flow rate of 1 ml/min of the mobile phase in the HPLC capillary system. PA1 2. Using suitable syringes, also aggressive reagents which may cause damage by corrosion in conventional HPLC pumps can be applied without any problem. PA1 3. Furthermore, depending on the respective construction of the pump, various reagents can be introduced into the system either in parallel and/or sequentially using two or more pairs of syringes.
a, of the eluate PA2 b, of the mixture of eluate and reagent
Parameters 1, 2 and 3a are relevant in principle in all processes of HPLC analytics, though the problems related to the instrumentation can be regarded as optimized to a great extent. Parameter 3b, however, is unique to PCR analysis and represents until now an unresolved problem restricting the use and the potential advantages of PCR systems in a serious manner.
A variety of methods were proposed, therefore, to improve the handling of reagent addition. Reagents, for example, were introduced into the eluate using porous hollow fiber membranes, permitting the diffusion of the molecules required for the derivatization of the analyte (U.S. Pat. No. 4,448,691). This method, however, is not universally applicable; it can be used only under some very special circumstances. Customary, in contrast, is the use of pumps infusing the reagent into the effluent of the column via a special mixing device. In most cases only a reduced pulsation rather than a pulsation-free addition of the reagent is possible resulting in a substantial increase of the background noise. The extent of the pulsation-related noise depends on the intensity of the detector specific signal properties of the reagent/eluate mixture as well as on the difference of their individual signal intensities. It follows that the detection limit deteriorates with increasing intensity of the background signal of the fluid reaching the detector resulting in an amplification of the low frequency noise caused by the pulsations of the pump used to infuse the reagent.
The reason for the customary use of HPLC pumps with low pulsation properties is the fact that the infusion of the reagent into the small-diameter capillaries carrying the effluent from the column to the detector requires substantial forces because of the high internal pressure of the system. This pressure cannot be overcome by normal commercially available pulsation-free pumps; this is relevant in particular, if--as necessary in most instances--post-column reactors, further increasing internal resistance, are used to extend reaction times in order to optimize the derivatization reaction. An additional disadvantage of the standard HPLC-pumps is that their pulsating properties are optimized for flow rates which are substantially higher than those required for the infusion of the reagent; furthermore, not all parts of these pumps being exposed to the reagent fluid are chemically resistant to its various components.
Frequently it is attempted to smooth pulsations by insertion of conventional pulse dampers. This, however, is not very effective, because these dampers are designed to reduce pulsations under conditions of extremely high pressure, i.e. prior to entry of the mobile phase into the analytical column.
Pulsation-free syringe pumps, on the other hand, equipped with either high frequency stepping or synchronized motors, can be used for PCR purposes only under very few special circumstances, i.e. unusually low systemic pressure combined with a low demand for reagent fluid, thus allowing the use of small diameter syringes. Syringes, however, providing sufficient volume for universal PCR application and full-time (8 h) operation must have large plunger diameters and, hence, require the buildup of high linear forces; neither the performance of the motors nor the mechanical properties of the transmission devices of such syringe pumps are able to overcome the systemic pressure under these circumstances or, alternatively, are damaged irreversibly after only a short period of time.
In conclusion, there is a demand for a generally applicable device to carry out post-column derivatizations in HPLC analytics permitting a pulsation-free supplementation of the column effluent with the necessary reagents even if pressures at the point of entry to the HPLC capillary system exceed 10 bar (1 MPa). This device should be able to operated full-time (8 h working day); it should be simple in its construction, easy to handle, reliable and economic in its operation and reasonably priced.